Studies in the <b>Epithelial Systems Biology Laboratory</b> of the <b>Systems Biology Center</b> (NHLBI) are focused on developing and understanding of the mechanistic basis of vasopressin-mediated regulation of water transport across the epithelium of the renal collecting duct. To do this, we are using a systems biology approach. Specifically, we are addressing the scientific problem by investigating networks of proteins that carry out the relevant regulatory tasks in the cell rather than focusing on any single protein such as the vasopressin V2 receptor or the water channel, aquaporin-2. For this we are using methods of large scale biology, viz, protein mass spectrometry and deep sequencing of DNA, coupled with mathematical methods for analysis of the data. A large part of our efforts are in the area of bioengineering, both with regard to the large-scale biological methods deployed and the computational methods that are used to analyze the data. Much of our current work focuses on development of methods for quantitative tandem mass spectrometry applied to proteins. Guided by a generalized mathematical model of cell signaling processes, we are using quantitative protein mass spectrometry-based proteomics for large-scale assessment of changes in post-translational modifications (esp. phosphorylation), intracellular localization, abundance, and binding to other proteins in renal collecting duct cells. Because we have found that many of the commercially available tools for analysis of large-scale biological data are imperfect or not appropriate for the tasks at hand, we are developing custom Java- and C++-based software tools for the analysis of proteomic and array data, with the objective of sharing these tools with the community. Regulation of transport in the collecting duct also involves transcriptional regulation. The laboratory has added RNA-Seq and ChIP-Seq approaches to hone in on the regulatory molecules that mediate vasopressin-regulated transcriptional changes in the renal collecting duct. RNA-Seq in single microdissected renal tubules is allowing us to discover signaling mechanisms responsible for transcriptional changes in animal models of water balance disorders such as the syndrome of inappropriate antidiuresis and lithium-induced nephrogenic diabetes insipidus. After critical proteins and protein modifications in vasopressin signaling are identified with 'discovery' approaches, we will investigate their roles using genome editing (CRISPR-Cas9) to ablate expression and/or introduce mutations in cultured collecting duct cells and mice.